© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1075-1084 doi:10.1242/dev.100123
RESEARCH ARTICLE
Eph-Ephrin signaling and focal adhesion kinase regulate actomyosin-dependent apical constriction of ciliary band cells
ABSTRACT Apical constriction typically accompanies inward folding of an epithelial sheet. In recent years there has been progress in understanding mechanisms of apical constriction and their contribution to morphogenetic processes. Sea urchin embryos form a specialized region of ectoderm, the ciliary band, which is a strip of epithelium, three to five cells wide, encircling the oral ectoderm and functioning in larval swimming and feeding. Ciliary band cells exhibit distinctive apical-basal elongation, have narrow apices bearing a cilium, and are planar polarized, so that cilia beat away from the mouth. Here, we show that filamentous actin and phosphorylated myosin light chain are uniquely distributed in ciliary band cells. Inhibition of myosin phosphorylation or actin polymerization perturbs this distribution and blocks apical constriction. During ciliary band formation, Sp-Ephrin and Sp-Eph expression overlap in the presumptive ciliary band. Knockdown of Sp-Eph or Sp-Ephrin, or treatment with an Eph kinase inhibitor interferes with actomyosin networks, accumulation of phosphorylated FAK (pY397FAK), and apical constriction. The cytoplasmic domain of Sp-Eph, fused to GST and containing a single amino acid substitution reported as kinase dead, will pull down pY397FAK from embryo lysates. As well, pY397FAK colocalizes with Sp-Eph in a JNK-dependent, planar polarized manner on latitudinal apical junctions of the ciliary band and this polarization is dissociable from apical constriction. We propose that Sp-Eph and pY397FAK function together in an apical complex that is necessary for remodeling actomyosin to produce centripetal forces causing apical constriction. Morphogenesis of ciliary band cells is a unique example of apical constriction in which receptor-mediated cell shape change produces a strip of specialized tissue without an accompanying folding of epithelium. KEY WORDS: Morphogenesis, Apical constriction, Eph-ephrin signaling, Focal adhesion kinase, Planar cell polarity, Ciliary band, Sea urchin
INTRODUCTION
Apical constriction is a cellular shape change that reduces apical surface area, producing bottle-shaped cells. This process is fundamental to morphogenetic movements of cells and cellular sheets that are integral to embryogenesis. It occurs coordinately in specific cell groups and is crucial to events such as gastrulation (Sweeton et al., 1991), neurulation (Nagele and Lee, 1987) and formation of specialized epithelia (Pilot and Lecuit, 2005; Sawyer et al., 2010). The mechanisms for achieving apical constriction appear to vary between organisms and even cell types within an Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 3P6, Canada. *Author for correspondence ([email protected]) Received 17 June 2013; Accepted 18 December 2013
organism (Sawyer et al., 2010), therefore characterizing these complex regulatory mechanisms is crucial in developing a generalized understanding of morphogenesis. It is widely accepted that actin filaments interact with myosin II motor units to provide mechanical force for apical constriction, and live imaging reveals concomitant organizational changes of actomyosin networks in apically constricting cells (Hildebrand, 2005; Lee and Harland, 2007). One of the most elaborate models for apical constriction occurs during Drosophila ventral furrow formation where the process is coordinated by a subcellular, ratcheting mechanism that produces a series of local, pulsed contractions in the supracellular, actomyosin meshwork; incrementally reducing apical surface area (Martin et al., 2009). Although constriction of the apical surface occurs in pulses, contraction and cortical tension within the actomyosin network occur prior to, and in the absence of, apical surface area reduction. This implies a regulatory mechanism controlling transient linkage of tensioned actomyosin networks to contact zones on the membrane surface (Roh-Johnson et al., 2012). Echinoderm embryos have a distinctive ectodermal structure, the ciliary band. This region is a continuous, three- to five-cell wide, band of cells encircling the oral field and forming a boundary between oral and aboral ectoderm. The ciliary band, like most ectoderm, is specified in late cleavage as a consequence of TGFβ signaling (Angerer et al., 2000; Duboc et al., 2004). Nodal is expressed ventrally and establishes oral ectoderm whereas BMP2/4, also expressed ventrally, acts with BMP5/8 to specify aboral ectoderm (Lapraz et al., 2009; Ben-Tabou de Leon et al., 2013). A band of cells between the major ectoderm domains is protected from this signaling and adopts the default state of becoming ciliary band ectoderm (Saudemont et al., 2010; Bradham et al., 2009; Yaguchi et al., 2010). The presumptive ciliary band cells are distinguished by expression of Hnf6, a putative core element of the gene regulatory network that specifies ciliary band cells (Otim et al., 2004; Poustka et al., 2004). Each ciliary band cell bears a cilium and their coordinated action provides feeding and locomotory functions for the larva (Strathmann, 1971; Strathmann et al., 1972). The cilia normally beat with their power stroke directed away from the oral field (Strathmann, 2007) and are functionally polarized in the plane of the epithelium. In Strongylocentrotus purpuratus, the ciliary band is first apparent at ~60 hours of development (Strathmann, 1971) when cells apparently reduce their apical surface area and become bottle shaped (Burke, 1978). The mechanism of this shape change has not been explored. The Eph receptor tyrosine kinases and their Ephrin ligands constitute the largest class of receptor tyrosine kinases in vertebrates. They are cell-surface molecules with roles in diverse biological processes, although they typically function at the interface of patterning and morphogenesis. Eph and Ephrin function in adhesion and regulate cytoskeletal organization by influencing regulatory protein complexes (Wilkinson, 2000; Klein, 2012). These include 1075
Development
Oliver A. Krupke and Robert D. Burke*
RESEARCH ARTICLE
interaction with focal adhesion kinase (FAK) (Carter et al., 2002), a non-receptor tyrosine kinase that regulates many cellular functions and has long been identified as a cytoskeletal regulator (Arold, 2011). Recent data identify FAK as an effector of Eph-Ephrin signaling, remodeling the cytoskeleton through recruitment and activation of Src-family kinases (Thomas et al., 1998; Parri et al., 2007; Shi et al., 2009; Darie et al., 2011). Here, we describe ciliary band morphogenesis in the developing sea urchin embryo. The ciliary band forms in the embryonic region where ectodermal expression domains of Sp-Eph and Sp-Ephrin overlap. We show that apical constriction is independent of cell division, and lossof-function experiments indicate that actin, myosin, Eph-Ephrin signaling and FAK are necessary for apical constriction. We propose that Eph-Ephrin signaling in the ciliary band provides a proximate cue initiating formation of a planar polarized, FAK-containing complex that regulates of apical constriction in ciliary band cells. Apical constriction of ciliary band cells is a distinctive model in which there is no inward folding of epithelium. RESULTS Apical surface area of ciliary band cells
The shape of ciliary band cells suggests apical constriction may be a feature of their development and we investigated whether apical surface area of ciliary band cells decreases during ciliary band formation. Between 48 and 96 hours of development, the ectoderm is transformed from a uniform sheet of cells into clearly defined regions of oral, aboral and ciliary band (Fig. 1). At 48 hours there is no measurable difference in surface area between ciliary band
Development (2014) doi:10.1242/dev.100123
cells and non-ciliary band cells (Fig. 1A″,E). At 55 hours, Hnf6positive cells have a noticeable reduction in their surface area (not shown). Over the next 17 hours the surface area of ciliary band cells is reduced by roughly one half; the majority of this occurring between 60 and 72 hours (Fig. 1A″,B″,E). Reduction in surface area is largely completed after 96 hours (Fig. 1D″). When viewed in cross section, ciliary band cells change their shape from having almost equal width and depth (Fig. 1F) to bottle-shaped (Fig. 1F′). This shape change appears to be an important event in ciliary band formation and we focused on identifying the underlying mechanism. Change in surface area in the absence of cell division
To assess whether reduction in apical surface area of ciliary band cells is due to localized cytokinesis, we inhibited DNA polymerase at 60 hours using aphidicolin and cultured embryos in the presence of 5-ethynyl-2¢-deoxyuridine (EdU) to label newly synthesized DNA and confirm inhibition of cytokinesis as an indirect effect. After 72 hours development, ciliary band cells in control embryos reduce their apical surface area (Fig. 2A′) and incorporate EdU into their nuclei (Fig. 2A), indicating DNA synthesis and subsequent cell division. Although aphidicolin completely blocks DNA synthesis (Fig. 2B) and indirectly prevents cytokinesis, ciliary band cells in treated embryos also appear to apically constrict (Fig. 2B′). Apical surface area measurements of ciliary band cells in embryos in which cell division is blocked and control embryos are not significantly different (P=0.062; Fig. 2C), indicating that reduction of apical surface area occurs independently of cytokinesis.
Development
Fig. 1. Apical constriction of ciliary band cells in S. purpuratus during early embryonic development. (A-A″) At 48 hours, cells expressing the ciliary band marker, Hnf6 are not apically constricted compared with non-Hnf6-expressing cells. (B-B″) At 72 hours, Hnf6-expressing cells appear constricted compared with non-ciliary band cells and there is a noticeable boundary (B′, arrowheads and B″) between the ciliary band, aboral and oral ectoderm. (C-D″) At 96 hours, Hnf6-expressing cells form a band of tissue (arrowheads) with cells arranged in compact rows (arrows) that encircle the oral field. (E) Apical surface area of ciliary band cells measured from 50 to 72 hours, illustrating apical constriction (50 hours, n=1292 cells from 19 embryos; 55 hours, n=1032 cells from 16 embryos; 60 hours, n=1196 cells from 16 embryos; 65 hours, n=724 cells in ten embryos; 72 hours, n=1045 cells in 12 embryos). (F,F′) Constriction of apical cell surface (top of image) causes ciliary band cells to change their cross-sectional shape from oval at 48 hours (F) to bottle at 72 hours (F′). Scale bars: 10 μm (A-D″); 5 μm (F,F′).
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RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.100123
Fig. 2. Apical constriction is independent of cell division. All images are of 72 hour embryos. (A) Incorporation of EdU from 60 to 72 hours illustrates many ciliary band cells are in S phase during this interval. (A′,A″) Apical constriction is evident in untreated embryos. (B) Incorporation of EdU from 60 to 72 hours is blocked by adding 0.6 μM aphidicolin at 60 hours. (B′,B″) Apical constriction is evident in treated embryos. (C) Blocking cell division from 60-72 hours has no significant effect on apical constriction in ciliary band cells (aphidicolin, n=570 cells in 12 embryos; control, 1551 cells in 19 embryos). Scale bars: 10 μm.
interfering with actomyosin contractility would lead to a loss of apical constriction in ciliary band cells. We tested this using cytochalasin D (Miyoshi et al., 2006) to disrupt actin filaments (Fig. 4A-A″), or ML 7 (Uehara et al., 2008) to inhibit myosin light chain kinase (Fig. 4BB″). Cytochalasin D-treated embryos are rounded with loosely packed cells expressing Hnf6 (Fig. 4A) and distribution of actin and pS19MLC are perturbed (Fig. 4A′,A″). Specifically, pS19MLC occurs in circular structures in the ciliary band (Fig. 4A′, arrows and inset). Similarly, actin networks in the ciliary band are discontinuous and appear as hollow circles (Fig. 4A″, arrows and inset). Embryos treated with ML 7 are rounded and Hnf6-expressing cells are loosely packed (Fig. 4B). Latitudinal distribution of pS19Myo is irregular (Fig. 4B′, arrowheads and inset). Similarly, actin networks are irregular and Fig. 3. Cytoskeletal networks of actin and phospho-myosin (pS19MLC) undergo remodeling in ciliary band cells during apical constriction with no apparent changes in actomyosin of non-ciliary band cells. (A) At 35 hours, cell junctions are clear and Hnf6 is not yet expressed. (A′,A″) pS19MLC and actin localize primarily to apical junctions, with minimal amounts in the apical cortex (inset, arrows). (B) At 55 hours, presumptive ciliary band cells express Hnf6 and cell junctions are indicated with anti-aPKC antibody. (B′) The polarized distribution of pS19MLC is evident on latitudinal membranes of the ciliary band (inset, arrow). (B″) Ciliary band-specific remodeling of actin is evident in the apical cortex (inset, arrow). (C) At 72 hours, ciliary band cells have constricted apically. (C′) A continuous, supracellular network of pS19MLC is present in the ciliary band, characterized by polarized expression on latitudinal junctions (inset, arrow). (C″) Continuous actin filaments form a supracellular network in the ciliary band accompanied by formation of dense actin networks in apical cortices of ciliary band cells (inset, arrow). Scale bars: 10 μm.
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Development
Actin cytoskeleton of the ciliary band
At 35 hours, actin and phosphorylated myosin light chain (pS19MLC) are distributed uniformly around the apical margin of ciliary band cells and colocalize with apical junction components (Fig. 3A-A′″). Beginning at about 40 hours, actin and pS19MLC are not associated solely with cell junctions (Fig. 3B-B″) but are distributed throughout the apical cortex of ciliary band cells. At 72 hours, pS19MLC is in discontinuous patches at the cell periphery and actin is dispersed in strands and patches in the apical cortex of ciliary band cells (Fig. 3C-C″). This rearrangement suggests a role during apical surface area reduction in ciliary band cells. We hypothesized that the actomyosin network provides some of the mechanical force that contributes to apical constriction and that
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.100123
Fig. 4. Treatment with cytochalasin D or ML 7 disrupts cytoskeletal networks of actin and phospho-myosin (pS19MLC) and inhibits apical constriction. All images are of 72 hour embryos. (A-A″) Embryos exposed to 20 μM cytochalasin D at 48 hours fail to form a ciliary band and form rounded embryos that lack a clearly defined ciliary band. (A) Ciliary band cells are loosely packed. (A′) Random accumulations of pS19MLC appear on apical junctions of ciliary band cells (inset, arrows). (A″) Patchy actin is distributed randomly on all cell junctions throughout the ciliary band (inset, arrows). (B-B″) Treatment with 5 μM ML 7 at 48 hours disrupts ciliary band formation. (B) Embryos are ovoid and lack a clearly defined ciliary band. (B′) Distribution of pS19MLC is polarized to latitudinal membranes in some areas of the ciliary band (arrowheads). Aberrant, pS19MLC-containing structures form in the apical cortices of cells (inset, arrow). (B″) Dense actin networks are present in only some areas of the ciliary band and actin does not accumulate in apical cortices (inset, arrowhead). (C) Inhibitors of actin polymerization and myosin light chain kinase block apical constriction of ciliary band cells. Apical surface area of ciliary band cells in control embryos is reduced more than 50% from 48 to 72 hours (solid line; 48 hours, n=625 cells in ten embryos; 60 hours, n=747 cells in ten embryos; 72 hours, n=788 cells in ten embryos). Apical constriction is significantly reduced in ciliary band cells of embryos treated with the cytoskeletal inhibitor cytochalasin D (dotted line; 48 hours, n=625 cells in ten embryos; 60 hours, n=637 cells in ten embryos; 72 hours, n=639 cells in ten embryos) or ML-7 (dashed line; 48 hours, n=625 cells in ten embryos; 60 hours, n=654 cells in ten embryos; 72 hours, n=597 cells in ten embryos). Scale bars: 10 μm.
Ectodermal expression of Sp-Eph and Sp-Ephrin and interaction with FAK
Preliminary data indicated that Sp-Eph and Sp-Ephrin were expressed in ectoderm beginning at gastrulation. Further analysis using antibodies against pY379Eph and Sp-Eph indicated that Sp-Eph is expressed throughout the oral and ciliary band ectoderm (Fig. 5E,F) and becomes phosphorylated in the ciliary band during apical constriction (Fig. 5A-B″; ciliary band boundary to oral ectoderm marked by arrowheads, boundary to aboral ectoderm marked by arrows). In the ciliary band, beginning at 48 hours, SpEph is polarized in its distribution; it is most abundant on latitudinal membranes (Fig. 5E,F, arrows). This contrasts with the expression in the oral ectoderm, where Sp-Eph is expressed uniformly around the periphery of cells (Fig. 5F, asterisks). Antibodies against SpEphrin indicate that the protein is expressed on aboral ectoderm and the ciliary band (Fig. 5A,A′,B,B′). Expression of Sp-Ephrin could not be detected in the oral ectoderm during ciliary band formation (Fig. 5B,B′). The overlapping expression of Sp-Eph and Sp-Ephrin in the region in which the ciliary band is forming and the detection of the phosphorylated form of the Sp-Eph receptor suggests that SpEph is signaling in presumptive ciliary band cells. A ciliary band expression pattern similar to that of Sp-Eph is observed using pY397FAK antibody (Fig. 5C,E′,F′), and these colocalize during apical constriction (Fig. 5E″,F″) suggesting an interaction. Following these observations, we investigated interactions between Sp-Eph and pY397FAK in vitro using a GST-tagged, cytoplasmic domain (322-762) of native Sp-Eph (Fig. 5D). This 1078
protein failed to reliably pull down pY397FAK in epithelial cell lysate from 72-hour embryos (Fig. 5D, lower panel). Because kinase domains often bind substrates transiently, releasing them upon phosphorylation, we created a kinase-dead form of this construct as a substrate trap (Roose et al., 2005) and we found it pulls down pY397FAK (Fig. 5D, top panel). This indicates a potential physical interaction between Sp-Eph and pY397FAK in ciliary band cells. Eph-Ephrin signaling is necessary for apical constriction
To assess the role of Eph-Ephrin signaling during apical constriction, we blocked translation of Sp-Eph or Sp-Ephrin in the embryo by morpholino-substituted antisense oligonucleotide (MASO) injection (Fig. 6). For each protein, we injected two, nonoverlapping oligonucleotides to knock down expression with 42.4% (±16.4% s.e.m.) mean knockdown based on fluorescence intensity. When we knocked down Sp-Eph or Sp-Ephrin, a distinct ciliary band failed to form after 72 hours and embryos were ovoid in shape (Fig. 6A-B′). At 72 hours, actin and pS19MLC in Sp-Eph knockdown embryos appear in circular or crescent-shaped patches throughout the ciliary band (Fig. 6C-C′″). These patches appear restricted to individual cells for pS19MLC (Fig. 6C′, arrow and inset) and for actin (Fig. 6C″, arrow and inset) and do not form an interconnected, supracellular network as seen in untreated embryos (Fig. 3), indicating Sp-Eph is necessary for actomyosin reorganization during ciliary band formation. Furthermore, ciliary band cells in MASO-injected embryos appear larger than in control embryos (Fig. 6D-F) and when we quantified apical surface area, we found apical constriction is significantly reduced in knockdown embryos (P
RESEARCH ARTICLE
Eph-Ephrin signaling and focal adhesion kinase regulate actomyosin-dependent apical constriction of ciliary band cells
ABSTRACT Apical constriction typically accompanies inward folding of an epithelial sheet. In recent years there has been progress in understanding mechanisms of apical constriction and their contribution to morphogenetic processes. Sea urchin embryos form a specialized region of ectoderm, the ciliary band, which is a strip of epithelium, three to five cells wide, encircling the oral ectoderm and functioning in larval swimming and feeding. Ciliary band cells exhibit distinctive apical-basal elongation, have narrow apices bearing a cilium, and are planar polarized, so that cilia beat away from the mouth. Here, we show that filamentous actin and phosphorylated myosin light chain are uniquely distributed in ciliary band cells. Inhibition of myosin phosphorylation or actin polymerization perturbs this distribution and blocks apical constriction. During ciliary band formation, Sp-Ephrin and Sp-Eph expression overlap in the presumptive ciliary band. Knockdown of Sp-Eph or Sp-Ephrin, or treatment with an Eph kinase inhibitor interferes with actomyosin networks, accumulation of phosphorylated FAK (pY397FAK), and apical constriction. The cytoplasmic domain of Sp-Eph, fused to GST and containing a single amino acid substitution reported as kinase dead, will pull down pY397FAK from embryo lysates. As well, pY397FAK colocalizes with Sp-Eph in a JNK-dependent, planar polarized manner on latitudinal apical junctions of the ciliary band and this polarization is dissociable from apical constriction. We propose that Sp-Eph and pY397FAK function together in an apical complex that is necessary for remodeling actomyosin to produce centripetal forces causing apical constriction. Morphogenesis of ciliary band cells is a unique example of apical constriction in which receptor-mediated cell shape change produces a strip of specialized tissue without an accompanying folding of epithelium. KEY WORDS: Morphogenesis, Apical constriction, Eph-ephrin signaling, Focal adhesion kinase, Planar cell polarity, Ciliary band, Sea urchin
INTRODUCTION
Apical constriction is a cellular shape change that reduces apical surface area, producing bottle-shaped cells. This process is fundamental to morphogenetic movements of cells and cellular sheets that are integral to embryogenesis. It occurs coordinately in specific cell groups and is crucial to events such as gastrulation (Sweeton et al., 1991), neurulation (Nagele and Lee, 1987) and formation of specialized epithelia (Pilot and Lecuit, 2005; Sawyer et al., 2010). The mechanisms for achieving apical constriction appear to vary between organisms and even cell types within an Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 3P6, Canada. *Author for correspondence ([email protected]) Received 17 June 2013; Accepted 18 December 2013
organism (Sawyer et al., 2010), therefore characterizing these complex regulatory mechanisms is crucial in developing a generalized understanding of morphogenesis. It is widely accepted that actin filaments interact with myosin II motor units to provide mechanical force for apical constriction, and live imaging reveals concomitant organizational changes of actomyosin networks in apically constricting cells (Hildebrand, 2005; Lee and Harland, 2007). One of the most elaborate models for apical constriction occurs during Drosophila ventral furrow formation where the process is coordinated by a subcellular, ratcheting mechanism that produces a series of local, pulsed contractions in the supracellular, actomyosin meshwork; incrementally reducing apical surface area (Martin et al., 2009). Although constriction of the apical surface occurs in pulses, contraction and cortical tension within the actomyosin network occur prior to, and in the absence of, apical surface area reduction. This implies a regulatory mechanism controlling transient linkage of tensioned actomyosin networks to contact zones on the membrane surface (Roh-Johnson et al., 2012). Echinoderm embryos have a distinctive ectodermal structure, the ciliary band. This region is a continuous, three- to five-cell wide, band of cells encircling the oral field and forming a boundary between oral and aboral ectoderm. The ciliary band, like most ectoderm, is specified in late cleavage as a consequence of TGFβ signaling (Angerer et al., 2000; Duboc et al., 2004). Nodal is expressed ventrally and establishes oral ectoderm whereas BMP2/4, also expressed ventrally, acts with BMP5/8 to specify aboral ectoderm (Lapraz et al., 2009; Ben-Tabou de Leon et al., 2013). A band of cells between the major ectoderm domains is protected from this signaling and adopts the default state of becoming ciliary band ectoderm (Saudemont et al., 2010; Bradham et al., 2009; Yaguchi et al., 2010). The presumptive ciliary band cells are distinguished by expression of Hnf6, a putative core element of the gene regulatory network that specifies ciliary band cells (Otim et al., 2004; Poustka et al., 2004). Each ciliary band cell bears a cilium and their coordinated action provides feeding and locomotory functions for the larva (Strathmann, 1971; Strathmann et al., 1972). The cilia normally beat with their power stroke directed away from the oral field (Strathmann, 2007) and are functionally polarized in the plane of the epithelium. In Strongylocentrotus purpuratus, the ciliary band is first apparent at ~60 hours of development (Strathmann, 1971) when cells apparently reduce their apical surface area and become bottle shaped (Burke, 1978). The mechanism of this shape change has not been explored. The Eph receptor tyrosine kinases and their Ephrin ligands constitute the largest class of receptor tyrosine kinases in vertebrates. They are cell-surface molecules with roles in diverse biological processes, although they typically function at the interface of patterning and morphogenesis. Eph and Ephrin function in adhesion and regulate cytoskeletal organization by influencing regulatory protein complexes (Wilkinson, 2000; Klein, 2012). These include 1075
Development
Oliver A. Krupke and Robert D. Burke*
RESEARCH ARTICLE
interaction with focal adhesion kinase (FAK) (Carter et al., 2002), a non-receptor tyrosine kinase that regulates many cellular functions and has long been identified as a cytoskeletal regulator (Arold, 2011). Recent data identify FAK as an effector of Eph-Ephrin signaling, remodeling the cytoskeleton through recruitment and activation of Src-family kinases (Thomas et al., 1998; Parri et al., 2007; Shi et al., 2009; Darie et al., 2011). Here, we describe ciliary band morphogenesis in the developing sea urchin embryo. The ciliary band forms in the embryonic region where ectodermal expression domains of Sp-Eph and Sp-Ephrin overlap. We show that apical constriction is independent of cell division, and lossof-function experiments indicate that actin, myosin, Eph-Ephrin signaling and FAK are necessary for apical constriction. We propose that Eph-Ephrin signaling in the ciliary band provides a proximate cue initiating formation of a planar polarized, FAK-containing complex that regulates of apical constriction in ciliary band cells. Apical constriction of ciliary band cells is a distinctive model in which there is no inward folding of epithelium. RESULTS Apical surface area of ciliary band cells
The shape of ciliary band cells suggests apical constriction may be a feature of their development and we investigated whether apical surface area of ciliary band cells decreases during ciliary band formation. Between 48 and 96 hours of development, the ectoderm is transformed from a uniform sheet of cells into clearly defined regions of oral, aboral and ciliary band (Fig. 1). At 48 hours there is no measurable difference in surface area between ciliary band
Development (2014) doi:10.1242/dev.100123
cells and non-ciliary band cells (Fig. 1A″,E). At 55 hours, Hnf6positive cells have a noticeable reduction in their surface area (not shown). Over the next 17 hours the surface area of ciliary band cells is reduced by roughly one half; the majority of this occurring between 60 and 72 hours (Fig. 1A″,B″,E). Reduction in surface area is largely completed after 96 hours (Fig. 1D″). When viewed in cross section, ciliary band cells change their shape from having almost equal width and depth (Fig. 1F) to bottle-shaped (Fig. 1F′). This shape change appears to be an important event in ciliary band formation and we focused on identifying the underlying mechanism. Change in surface area in the absence of cell division
To assess whether reduction in apical surface area of ciliary band cells is due to localized cytokinesis, we inhibited DNA polymerase at 60 hours using aphidicolin and cultured embryos in the presence of 5-ethynyl-2¢-deoxyuridine (EdU) to label newly synthesized DNA and confirm inhibition of cytokinesis as an indirect effect. After 72 hours development, ciliary band cells in control embryos reduce their apical surface area (Fig. 2A′) and incorporate EdU into their nuclei (Fig. 2A), indicating DNA synthesis and subsequent cell division. Although aphidicolin completely blocks DNA synthesis (Fig. 2B) and indirectly prevents cytokinesis, ciliary band cells in treated embryos also appear to apically constrict (Fig. 2B′). Apical surface area measurements of ciliary band cells in embryos in which cell division is blocked and control embryos are not significantly different (P=0.062; Fig. 2C), indicating that reduction of apical surface area occurs independently of cytokinesis.
Development
Fig. 1. Apical constriction of ciliary band cells in S. purpuratus during early embryonic development. (A-A″) At 48 hours, cells expressing the ciliary band marker, Hnf6 are not apically constricted compared with non-Hnf6-expressing cells. (B-B″) At 72 hours, Hnf6-expressing cells appear constricted compared with non-ciliary band cells and there is a noticeable boundary (B′, arrowheads and B″) between the ciliary band, aboral and oral ectoderm. (C-D″) At 96 hours, Hnf6-expressing cells form a band of tissue (arrowheads) with cells arranged in compact rows (arrows) that encircle the oral field. (E) Apical surface area of ciliary band cells measured from 50 to 72 hours, illustrating apical constriction (50 hours, n=1292 cells from 19 embryos; 55 hours, n=1032 cells from 16 embryos; 60 hours, n=1196 cells from 16 embryos; 65 hours, n=724 cells in ten embryos; 72 hours, n=1045 cells in 12 embryos). (F,F′) Constriction of apical cell surface (top of image) causes ciliary band cells to change their cross-sectional shape from oval at 48 hours (F) to bottle at 72 hours (F′). Scale bars: 10 μm (A-D″); 5 μm (F,F′).
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RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.100123
Fig. 2. Apical constriction is independent of cell division. All images are of 72 hour embryos. (A) Incorporation of EdU from 60 to 72 hours illustrates many ciliary band cells are in S phase during this interval. (A′,A″) Apical constriction is evident in untreated embryos. (B) Incorporation of EdU from 60 to 72 hours is blocked by adding 0.6 μM aphidicolin at 60 hours. (B′,B″) Apical constriction is evident in treated embryos. (C) Blocking cell division from 60-72 hours has no significant effect on apical constriction in ciliary band cells (aphidicolin, n=570 cells in 12 embryos; control, 1551 cells in 19 embryos). Scale bars: 10 μm.
interfering with actomyosin contractility would lead to a loss of apical constriction in ciliary band cells. We tested this using cytochalasin D (Miyoshi et al., 2006) to disrupt actin filaments (Fig. 4A-A″), or ML 7 (Uehara et al., 2008) to inhibit myosin light chain kinase (Fig. 4BB″). Cytochalasin D-treated embryos are rounded with loosely packed cells expressing Hnf6 (Fig. 4A) and distribution of actin and pS19MLC are perturbed (Fig. 4A′,A″). Specifically, pS19MLC occurs in circular structures in the ciliary band (Fig. 4A′, arrows and inset). Similarly, actin networks in the ciliary band are discontinuous and appear as hollow circles (Fig. 4A″, arrows and inset). Embryos treated with ML 7 are rounded and Hnf6-expressing cells are loosely packed (Fig. 4B). Latitudinal distribution of pS19Myo is irregular (Fig. 4B′, arrowheads and inset). Similarly, actin networks are irregular and Fig. 3. Cytoskeletal networks of actin and phospho-myosin (pS19MLC) undergo remodeling in ciliary band cells during apical constriction with no apparent changes in actomyosin of non-ciliary band cells. (A) At 35 hours, cell junctions are clear and Hnf6 is not yet expressed. (A′,A″) pS19MLC and actin localize primarily to apical junctions, with minimal amounts in the apical cortex (inset, arrows). (B) At 55 hours, presumptive ciliary band cells express Hnf6 and cell junctions are indicated with anti-aPKC antibody. (B′) The polarized distribution of pS19MLC is evident on latitudinal membranes of the ciliary band (inset, arrow). (B″) Ciliary band-specific remodeling of actin is evident in the apical cortex (inset, arrow). (C) At 72 hours, ciliary band cells have constricted apically. (C′) A continuous, supracellular network of pS19MLC is present in the ciliary band, characterized by polarized expression on latitudinal junctions (inset, arrow). (C″) Continuous actin filaments form a supracellular network in the ciliary band accompanied by formation of dense actin networks in apical cortices of ciliary band cells (inset, arrow). Scale bars: 10 μm.
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Development
Actin cytoskeleton of the ciliary band
At 35 hours, actin and phosphorylated myosin light chain (pS19MLC) are distributed uniformly around the apical margin of ciliary band cells and colocalize with apical junction components (Fig. 3A-A′″). Beginning at about 40 hours, actin and pS19MLC are not associated solely with cell junctions (Fig. 3B-B″) but are distributed throughout the apical cortex of ciliary band cells. At 72 hours, pS19MLC is in discontinuous patches at the cell periphery and actin is dispersed in strands and patches in the apical cortex of ciliary band cells (Fig. 3C-C″). This rearrangement suggests a role during apical surface area reduction in ciliary band cells. We hypothesized that the actomyosin network provides some of the mechanical force that contributes to apical constriction and that
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.100123
Fig. 4. Treatment with cytochalasin D or ML 7 disrupts cytoskeletal networks of actin and phospho-myosin (pS19MLC) and inhibits apical constriction. All images are of 72 hour embryos. (A-A″) Embryos exposed to 20 μM cytochalasin D at 48 hours fail to form a ciliary band and form rounded embryos that lack a clearly defined ciliary band. (A) Ciliary band cells are loosely packed. (A′) Random accumulations of pS19MLC appear on apical junctions of ciliary band cells (inset, arrows). (A″) Patchy actin is distributed randomly on all cell junctions throughout the ciliary band (inset, arrows). (B-B″) Treatment with 5 μM ML 7 at 48 hours disrupts ciliary band formation. (B) Embryos are ovoid and lack a clearly defined ciliary band. (B′) Distribution of pS19MLC is polarized to latitudinal membranes in some areas of the ciliary band (arrowheads). Aberrant, pS19MLC-containing structures form in the apical cortices of cells (inset, arrow). (B″) Dense actin networks are present in only some areas of the ciliary band and actin does not accumulate in apical cortices (inset, arrowhead). (C) Inhibitors of actin polymerization and myosin light chain kinase block apical constriction of ciliary band cells. Apical surface area of ciliary band cells in control embryos is reduced more than 50% from 48 to 72 hours (solid line; 48 hours, n=625 cells in ten embryos; 60 hours, n=747 cells in ten embryos; 72 hours, n=788 cells in ten embryos). Apical constriction is significantly reduced in ciliary band cells of embryos treated with the cytoskeletal inhibitor cytochalasin D (dotted line; 48 hours, n=625 cells in ten embryos; 60 hours, n=637 cells in ten embryos; 72 hours, n=639 cells in ten embryos) or ML-7 (dashed line; 48 hours, n=625 cells in ten embryos; 60 hours, n=654 cells in ten embryos; 72 hours, n=597 cells in ten embryos). Scale bars: 10 μm.
Ectodermal expression of Sp-Eph and Sp-Ephrin and interaction with FAK
Preliminary data indicated that Sp-Eph and Sp-Ephrin were expressed in ectoderm beginning at gastrulation. Further analysis using antibodies against pY379Eph and Sp-Eph indicated that Sp-Eph is expressed throughout the oral and ciliary band ectoderm (Fig. 5E,F) and becomes phosphorylated in the ciliary band during apical constriction (Fig. 5A-B″; ciliary band boundary to oral ectoderm marked by arrowheads, boundary to aboral ectoderm marked by arrows). In the ciliary band, beginning at 48 hours, SpEph is polarized in its distribution; it is most abundant on latitudinal membranes (Fig. 5E,F, arrows). This contrasts with the expression in the oral ectoderm, where Sp-Eph is expressed uniformly around the periphery of cells (Fig. 5F, asterisks). Antibodies against SpEphrin indicate that the protein is expressed on aboral ectoderm and the ciliary band (Fig. 5A,A′,B,B′). Expression of Sp-Ephrin could not be detected in the oral ectoderm during ciliary band formation (Fig. 5B,B′). The overlapping expression of Sp-Eph and Sp-Ephrin in the region in which the ciliary band is forming and the detection of the phosphorylated form of the Sp-Eph receptor suggests that SpEph is signaling in presumptive ciliary band cells. A ciliary band expression pattern similar to that of Sp-Eph is observed using pY397FAK antibody (Fig. 5C,E′,F′), and these colocalize during apical constriction (Fig. 5E″,F″) suggesting an interaction. Following these observations, we investigated interactions between Sp-Eph and pY397FAK in vitro using a GST-tagged, cytoplasmic domain (322-762) of native Sp-Eph (Fig. 5D). This 1078
protein failed to reliably pull down pY397FAK in epithelial cell lysate from 72-hour embryos (Fig. 5D, lower panel). Because kinase domains often bind substrates transiently, releasing them upon phosphorylation, we created a kinase-dead form of this construct as a substrate trap (Roose et al., 2005) and we found it pulls down pY397FAK (Fig. 5D, top panel). This indicates a potential physical interaction between Sp-Eph and pY397FAK in ciliary band cells. Eph-Ephrin signaling is necessary for apical constriction
To assess the role of Eph-Ephrin signaling during apical constriction, we blocked translation of Sp-Eph or Sp-Ephrin in the embryo by morpholino-substituted antisense oligonucleotide (MASO) injection (Fig. 6). For each protein, we injected two, nonoverlapping oligonucleotides to knock down expression with 42.4% (±16.4% s.e.m.) mean knockdown based on fluorescence intensity. When we knocked down Sp-Eph or Sp-Ephrin, a distinct ciliary band failed to form after 72 hours and embryos were ovoid in shape (Fig. 6A-B′). At 72 hours, actin and pS19MLC in Sp-Eph knockdown embryos appear in circular or crescent-shaped patches throughout the ciliary band (Fig. 6C-C′″). These patches appear restricted to individual cells for pS19MLC (Fig. 6C′, arrow and inset) and for actin (Fig. 6C″, arrow and inset) and do not form an interconnected, supracellular network as seen in untreated embryos (Fig. 3), indicating Sp-Eph is necessary for actomyosin reorganization during ciliary band formation. Furthermore, ciliary band cells in MASO-injected embryos appear larger than in control embryos (Fig. 6D-F) and when we quantified apical surface area, we found apical constriction is significantly reduced in knockdown embryos (P
